Cellular electrophysiological basis for cardiac arrhythmia is due to blockage of ion channels, resulting in delay of repolarization and prolongation of QT interval, characteristics of ECG with longer interval between Q and T points (Keating and Sanguinetti. 2001; Roden et al. 2002). Because it is the target of most non-cardiac drugs that cause cardiac arrhythmia (Abriel et al. 2004; Joshi et al. 2004), special attention is paid to hERG (human ether-à-go-go-related gene) which encodes for the pore-forming α subunit of the rapidly activating delayed rectifier potassium channel. The channel is responsible for the repolarizing potassium current which is distinguished from other current by strong inward rectification. Inhibition of this channel, either by exogenous compounds or due to genetic mutation, increases the duration of repolarization of action potential in cardiac myocytes, resulting in cardiac arrhythmias. Nevertheless, cardiac arrhythmia is not only due to the malfunction of hERG but also other ion channels, such as sodium channels and calcium channels. Thus, emphasis is put on the preclinical screening for new therapeutic compounds with any unexpected cardiac toxic effects by highly sensitive and specific experimental models (Joshi et al. 2004).
Traditionally, patch clamp electrophysiology is regarded as the “gold standard” for measuring of ion channel activity (Fermini and Fossa. 2003). Patch clamp allows for the direct and real-time monitoring of ion channel activity in the administration of testing compounds in whole-cell mode. However, it is a low-throughput methodology and requires highly skillful operators. Although other advanced screening technologies have been developed recently to improve the degree of throughput, all these technologies are based on in vitro cell culture models. The disadvantage of using in vitro model is that complex physiological environment that occurs in vivo cannot be modeled in an in vitro system. Some biological questions of cardiac electrophysiology cannot simply be addressed by in vitro assays. In this case, in vivo models have been applied, including Guinea pig, dog, and primate. ECG is continuously recorded over a range of increasing doses in anaesthetized animal to detect the occurrence of any cardiac arrhythmia. Moreover, they are not widely used due to the ethical issues and cost efficiency. Therefore, there is a need to develop a new technology with lower cost and higher efficiency.
Zebrafish has emerged as a model in developmental biology studies, toxicology studies as well as pharmaceutical studies. Zebrafish in vivo bioassay combines the advantages of high throughput, as compared to mammalian in vivo assays, and high relevance, as compared to in vitro assays. Recently, two articles have been published demonstrating similar physiological responses of zebrafish to well-know compounds that induce cardiac arrhythmia in humans (Langheinrich et al. 2003; Milan et al. 2003). Furthermore, the ortholog of hERG was cloned and showed high similarity in protein sequence in the pore region and the cyclic-nucleotide binding region to which some cardiac toxic compounds bind (Langheinrich et al. 2003). In addition, mutation in zERG exhibits similar phenotype of cardiac arrhythmia as in human. These results suggest zebrafish may be used as a model for screening compounds with cardiac toxicity.
Methods have been developed to measure cardiac function in zebrafish. However, they are usually low throughput, time consuming and labor intensive. The simplest method is to use a stopwatch to count number of heart beat per minute under conventional light microscope or stereomicroscope (Langheinrich et al. 2003). An image analysis method of digital movie of heart has been developed (Milan et al. 2003). Average pixel intensity of a particular region of heart was measured. Fast Fourier transform of these data was performed to determine the heart rate. Beside heart rate, other cardiac parameters, e.g. cardiac output, hemodynamics and electrical properties, have been developed (reviewed by (Schwerte and Fritsche. 2003)). Cardiac output, an important parameter of cardiac physiology, can be determined non-invasively in transparent zebrafish embryos by calculation of the ventricular volume during cardiac cycle by filming the beating ventricle. The formula of volume calculation requires the length of axes of ventricle which can be obtained by outlining the ventricle manually or automatically with the assistance of a computer program. Hemodynamics as determined by blood pressure can be measured using servo-null micropressure system. In the system, a glass capillary filled with a NaCl solution of high concentration is inserted into the blood vessels of interest using a micromanipulator. Pressure change in the blood vessel will move the interface between the plasma and NaCl solution, resulting in the change of electrical resistance of the electrode inside the glass capillary. Recently, methodology for measuring electrical properties in zebrafish embryos has been developed (Forouhar et al. 2004). In the methodology, five day old embryos are mounted on their dorso-ventral orientation and two electrodes are positioned using a micromanipulator, one on the body surface outside the heart and another reference electrode in the surrounding solution. However, these methods are also time-consuming and labor intensive, particularly in sample preparation steps, making them not suitable for high throughput study.